This disclosure relates to compositions, kits, and methods for the synthesis and/or detection of nucleic acids.
For many medical, diagnostic, and forensic applications, amplification of a particular DNA sequence is essential to allow its detection in, or isolation from, a sample in which it is present in very low amounts. More recently, in vitro amplification of specific genes has provided powerful and less costly means to facilitate the production of therapeutic proteins by molecular biological techniques, and may have applications in genetic therapy as well.
The polymerase chain reaction (PCR) technique is disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188. The PCR method is also described in Saiki et al., 1985, Science 230:1350. In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A repetitive series of reaction steps involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5′ ends of the primers. PCR is capable of producing a selective enrichment of a specific DNA sequence by a factor of 109.
Commercially available PCR master mixes improve the efficiency and reduce the errors associated with the assembly of large number of PCR reactions required for high-throughput analysis. These master mixes contain a combination of reagents that will be common to all PCR reactions. For example, the master mix may contain a buffer, a salt such as MgCl2, deoxynucleoside triphosphates (dNTPs), and a thermostable DNA polymerase. Each well would contain the common master mix and a specific target nucleic acid and primer pair. Typically, master mixes are manufactured and distributed as concentrated solutions or lyophilized powders which are subsequently diluted or dissolved when final reactions are assembled.
For accurate analysis, PCR master mixes should provide reliable, robust, and reproducible PCR results. Further, PCR master mixes should allow for detection of low copy number target nucleic acids. The PCR master mix should also allow for fast PCR reaction cycles to allow rapid screening of nucleic acids (e.g., DNAs and cDNAs). In addition, the PCR master mix should also be stable on the bench top at ambient or room temperature so that the PCR reactions need not be amplified immediately after assembly.
Sources for nucleic acid samples include, but are not limited to, for example clothing, soil, paper, metal surfaces, air, water, plant parts, as well as human and/or animal skin, hair, blood, serum, feces, milk, saliva, urine, and/or other secretory fluids. These sources may also contain compounds that inhibit PCR amplification.
Accordingly, there is a need to identify agents that block or reduce the inhibition of PCR amplification by components found in sources for nucleic acid samples.